NUC-1031 is a prodrug of gemcitabine developed by NuCana BioMed Ltd., and is currently in Phase II clinical trials for the treatment of cancers such as advanced solid tumors, pancreatic cancer and breast cancer. The CAS of NUC-1031 is 840506-29-8 and NUC-1031 has a structure represented by the following formula 1, including two single isomers, Rp-1 and Sp-1:

On page 70 in the description of WO2005012327 are disclosed the specific structure of NUC-1031 and the preparation method thereof, the method comprising, specifically, in the presence of NMI, reacting gemcitabine with benzyl-(benzoyl-L-alanine)-chlorophosphate at a molar ratio of 1:3 for 2 hours, with THF/pyridine as the solvent, to give a crude product, which is then subjected to separation and purification on a silica gel column, and eluted with dichloromethane/methanol (95:5) to give a foamy solid with a yield of only 16%.
WO2014076490A1 discloses the following method for preparing NUC-1031,

3′-Boc-protected gemcitabine (100 mg) is reacted with 2 mol equivalents of phenyl(benzyl-L-alanine)chlorophosphate (150 mg), with 0.5 mol equivalents of tris(acetylacetone)Fe(III) (56 mg) as the catalyst, 1.5 mol equivalents of DIPEA (55 μL) as the base, and 10 ml of THF as the solvent; the reaction is conducted at room temperature under the protection of nitrogen gas for 24 hours with a yield of 45%, wherein the ratio of the isomers, i.e., Rp:Sp is 3:1.
A literature entitled “Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development” (Journal of Medicinal Chemistry, Volume 57, Issue 4, Pages 1531-1542) reported a method for preparing NUC-1031 by deprotecting the hydroxyl group of the compound 5f represented by the following formula in the presence of TFA with dichloromethane as the solvent and finally by purification on a silica gel column, the yield is 70%, the contents of the isomers are 48% and 52%, respectively.

At present, the preparation method of the single isomer of NUC-1031 has not been reported in the prior art. Since the two enantiomers of the chiral P in the molecular structure of NUC-1031 are very similar in structure and polarity, it becomes very difficult to isolate a high-purity single isomer from the racemic mixture of NUC-1031, and in particular, it is even more difficult to balance purity and yield simultaneously in the purification process. The inventors have attempted to purify NUC-1031 by various conventional methods in the art such as crystallization, silica gel column chromatography, as well as reversed phase C18 silica gel-bonded preparative chromatography, normal phase preparative chromatography with spherical silica based packing, and chiral chromatographic separation, but all of them can hardly isolate a single isomer with a purity of not less than 90%.
It is known in the art that usually when a chiral compound is used as a drug, the relationship between the configuration and the pharmacological effect of the isomer can be divided into the following scenarios: (1) the pharmacological effect of the drug is completely or mainly generated by one of the enantiomers, for example, (S)-naproxen has an analgesic effect 35 times stronger than that of the R isomer; (2) the two isomers have completely opposite pharmacological effects, for example, the dextrorotatory isomer of Picenadol is an opiate receptor agonist, while the levorotatory isomer thereof is an opiate receptor antagonist; (3) an isomer arises serious adverse reactions, for example, the emetic reaction of the anthelmintic tetramisole is caused by the dextrorotatory isomer thereof; (4) one pharmacological effect has high stereoselectivity, while the other pharmacological effects have low or no stereoselectivity, for example, addiction to the antitussive methorphan is mainly caused by the levorotatory isomer thereof, but in terms of the antitussive effect, the dextrorotatory isomer and the levorotatory isomer have the same intensity; and (5) the two enantiomers have different pharmacological effects, but the combination use is beneficial, for example, the dextrorotatory isomer of the antihypertensive nehivolol is a β-receptor blocker, while the levorotatory isomer thereof can reduce peripheral vascular resistance and has protective effect for heart.
It is known that NUC-1031 molecule contains many chiral atoms, and isolating single isomers from NUC-1031, studying their biological activities respectively, and studying the pharmacological effects, toxic side effects or adverse reactions of NUC-1031 to develop similar drugs with better activity and less toxic side effects will be of great significance. Preparing high-purity single isomers that meet the needs of clinical research and screening the single isomer that plays an important role in the pharmacological activity of NUC-1031 are still problems that need to be solved in the art at present.